In scanning microscopy (raster microscopy), a specimen is illuminated with a light beam so that the reflection light or fluorescent light emitted by the specimen can be observed. The focus of the illuminating light beam is moved in an object plane by means of a controllable beam deflector, generally by tilting two mirrors, whereby the deflection axes are usually positioned perpendicular to each other, so that one mirror deflects in the x-direction while the other deflects in the y-direction. The mirrors are tilted, for example, employing galvanometric actuating elements. The power of the light coming from the object is measured as a function of the position of the scanning beam. Normally, the actuating elements are equipped with sensors to ascertain the actual position of the mirror. Aside from these so-called beam-scanning methods, scanning microscopes with a spatially stationary illuminating light beam are likewise known, in which a fine-positioning stage is used to move the specimen for scanning purposes. These scanning microscopes are known as object-scanning microscopes.
Especially in the field of confocal scanning microscopy, an object is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light from the source is focused onto a pinhole diaphragm (the so-called excitation diaphragm), a beam splitter, a beam deflector to control the beam, a microscope optical system, a detection diaphragm and the detectors for picking up the detection or fluorescent light. The illumination light is coupled in via a beam splitter. Via the beam deflector, the fluorescent or reflection light coming from the object returns to the beam splitter, passes through it and is subsequently focused onto the detection diaphragm behind which the detectors are located. Detection light that does not stem directly from the focus region takes a different light path and does not pass the detection diaphragm, so that point information is obtained that yields a three-dimensional image as a result of the sequential scanning of the object. For the most part, a three-dimensional image is attained by means of layer-by-layer image data acquisition.
German publication DE 44 16 558 discloses an arrangement to increase the resolving power for fluorescence applications. Here, the lateral edge areas of the focus volume of the excitation light beam are illuminated with a light beam having a different wavelength, the so-called stimulation light beam, which is emitted by a second laser, in order for the areas of the specimen excited by the light of the first laser to be returned to their ground state, said areas now having been stimulated. Only the light that is spontaneously emitted from the areas that are not illuminated by the second laser is detected, so that altogether, an improvement in the resolution is achieved. The designation STED (Stimulated Emission Depletion) has been given to this method.
For instance, U.S. patent application 2002/0167724 or U.S. Pat. No. 6,667,830 disclose a variant of the STED technique in which the areas of the specimen excited by the light of the first laser are at first further excited, namely, into a third state, with the light of the second laser. This variant, which has become known by the name “up-conversion”, achieves an increase in the resolution in a manner equivalent to the variant involving the directly stimulated depletion into the ground state.
German patent application DE 100 12 462 A1 discloses a device for illuminating an object, preferably in confocal fluorescence scanning microscopy, with an illuminating beam path of a light source and at least one additional illuminating beam path of an additional light source, whereby the illuminating beam paths can be superimposed over each other, at least partially. This device is aimed at simplifying the adjustment as well as reducing the number of optical components in the illuminating beam path and it is characterized in that at least one optical component is arranged in at least one of the illuminating beam paths, whereby the optical properties of the component can be influenced or changed in such a way that the illumination pattern of the illuminating beam path changes its shape in the area of the object. In this context, the optical component can be configured, for instance, as a round phase-retarding plate whose diameter is smaller than the diameter of the beam and that is consequently fully illuminated. The term “phase-retarding plate” is used for an optical component that brings about a location-dependent phase retardation of the light that passes through said phase-retarding plate. The phase-retarding plate is arranged in the beam path of the illuminating beam that triggers a stimulated emission and, with an appropriate structure, generates a hollow focus that allows an improvement in the resolution, both laterally and axially. A preferred embodiment of a phase-retarding plate consists of a substrate onto which one or more layers of a phase-retarding material (for example, MgF2) are applied locally in certain areas. If the thickness of the layers and the size of the layer areas are selected in such a way that half of the total light amplitude in the pupil of the microscope optical system has a phase retardation of λ/2 relative to the other half of the light amplitude, then the focused wave front generates destructive interference in the focus of the microscope optical system (objective). The resultant PSF (Point Spread Function) thus has a minimum of the focus center.
The use of phase-retarding plates in STED microscopy is also cited, for example, in the following publications: Proc. Natl. Acad. Sci. U.S.A., Vol. 97, p. 8206 to 8210, 2000; Appl. Phys. Lett., Vol. 82, No. 18, p. 3125 to 3127, 2003; Phys. Rev. Lett., Vol. 88, p. 163901-1 to 163901-4, 2002; Phys. Rev. E, Vol. 64, p. 066613-1 to 066613-9, 2001.
Instead of conventional phase-retarding plates, it is also possible to employ LCDs or programmable light modulators.
A resolution increase in the direction of the optical axis can be achieved by means of a double-objective array (4Pi-array) in the manner described in European patent specification EP 0 491 289 bearing the title: “Doppelkonfokales Rastermikroskop” [“Double confocal scanning microscope”]. The excitation light coming from the illumination system is split into two partial beams that simultaneously illuminate the specimen from opposite directions through two mirror-symmetrical objectives. The two objectives are arranged on different sides of the object plane they share. As a result of this interferometric illumination, an interference pattern having a main maximum and several secondary maxima is formed in the object point in the case of constructive interference. If only the light of the excitation interferes, one speaks of 4Pi microscopy of Type A, while Type C refers to simultaneous interference of the detection light. Owing to the interferometric illumination, a higher axial resolution can be achieved with this double confocal scanning microscope than with a conventional scanning microscope.
The combination of STED and a double confocal array permits both a lateral and an axial improvement of the resolution.
In a special combination of an STED and a double confocal array, namely, the STED-4Pi microscope, a double confocal array of the stimulation light beam serves to generate a destructive interference in the focus center. Consequently, a stimulated depletion is restricted to the axial focus edge (Phys. Rev. Lett., Vol. 88, p. 1639901-1 to 163901-4, 2002).
It has been found that, in order to achieve the best possible resolving power, the optical component has to be positioned and adjusted very precisely, preferably in the beam path of the stimulation light beam. A drawback of this approach is that the optical component has to be readjusted and adapted to the structural and optical properties of the new objective every time the objective is changed.